Project Summary/Abstract Intrinsic and acquired drug resistance of medically relevant microorganisms poses a grave threat to human health and has enormous economic consequences worldwide. Fungal pathogens present a particular challenge because they are eukaryotes and share many of the same biological processes as the human hosts they infect. Among the most pervasive fungal pathogens are species of Cryptococcus, which cause over 600,000 deaths per year. Cryptococcal meningitis, the major clinical manifestation of the disease, has a 100% mortality rate if left untreated. Even with the best available therapies, mortality rates remain high at 35-40% because the number of drug classes that have distinct targets in fungi is very limited and the usefulness of current antifungal drugs is compromised by either dose-limiting host toxicity or the frequent emergence of high- grade resistance. New, non-cross-reactive targets for therapeutic intervention are urgently needed. In previous work, we discovered that that the molecular chaperone Hsp90 regulates drug resistance and virulence in species of the fungi Candida and Aspergillus. Targeting Hsp90 in these pathogens promises to provide a powerful strategy to enhance the efficacy of antifungal drugs and abrogate drug resistance, but the role of Hsp90 in pathogenic cryptococcal species remains unexplored. The ?druggability? of Hsp90 has been well established by the many small molecules targeting this protein for the treatment of human cancers. The poor antifungal activity and likely toxicity of currently available drugs in the setting of fungal infection, however, demand the development of fungal-selective Hsp90 inhibitors. To pursue the goal of fungal selectivity, our interdisciplinary team solved the structure of the N-terminal domain of Candida albicans Hsp90, and identified a pocket in the nucleotide-binding region that is larger than its human counterpart and is conserved in Cryptococcus. Guided by this insight, we designed, synthesized and characterized two lead inhibitors of fungal Hsp90 with >10-fold selectivity relative to the human protein. Now, leveraging the novel chemistry and structure-based design approach we have developed, we will use our complementary expertise in fungal biology (Cowen), chemistry (Brown), and pharmacology/experimental therapeutics (Whitesell) to pursue structure activity relationship (SAR) studies on libraries of additional analogs and generate selective drug-like probes. These will be used in a powerful combination of genetic and pharmacological approaches to dissect Hsp90's role in the drug resistance and virulence of Cryptococcus. In addition to the important basic insights that will be obtained, our results are likely to impact the treatment of invasive fungal infections in the near future by providing promising leads for the development of drug candidates that operate in a completely unexploited target space.